This invention relates to tumor-inhibiting conjugates of proteins and polymers consisting of a suitable carrier system and cytostatic compounds. Further, the invention relates to methods for the production of such conjugates and the use of these. Immuno-conjugates or conjugates of protein or of polymer are compounds which consist of a suitable carrier substance, such as, for example, an antibody, a growth factor, a structure similar to hormones or peptides, a protein or a polymer, and one or more cytotoxic active substances such as, for example, cytostatics, toxins or radioactive isotopes. The carrier substances have, as a rule, the characteristic of preferably accumulating in the tumor tissue, so that in this way also the active substance bound to the carrier substance accumulates in the tumor tissue and thus a selective anti-tumor therapy is achieved. Chemoimmuno-conjugates are conjugates of carrier substances and cytostatic compounds, wherein the carrier, as a rule, is an antibody.
The cytostatics currently used against cancers have a series of strong systemic side-effects and do not exhibit accumulation in the tumor tissue, so that new derivatives and formulations are being researched which make selective anti-tumor therapy possible. For this purpose. chemoimmuno-conjugates or conjugates of proteins or of polymers consisting of one suitable carrier substance or cytostatics are being developed.
As carrier substances, among others, antibodies, growth factors, serum proteins, structures similar to hormones or peptides, or polymers are considered, for which, as a rule, an accumulation in the tumor tissue is known (Mxc3xa4gerstxc3xa4dt, M.: Antibody Conjugates and Malignant Disease, Library of Congress 1990: Chadwick, C. M.: Receptors in Tumour Biology, Cambridge University Press, 1984, Seymour, L. W. CRC Crit. Rev. Ther. Drug Carrier Sys. (1992), 9, 135-187; Maeda, H.; Matsumura, Y. CRC Crit. Rev. Ther. Drug Carrier Sys. (1989), 6, 193-210). The present invention comprises human serum transferrin and serum albumin as carrier proteins, of which the accumulation in the tumor tissue is documented (Ward, S. G. Taylor, R. C.: 1-54, in Metal-Based Drugs (Gielen, M. F. (Ed.)), Freund Publishing House Ltd, 1988; Sinn, H., Schrenk, H. H., Friedrich, A., Schilling, U. and Maier-Borst, W. (1990), Nucl. Med Biol. Vol. 17(8), 819-827) as well as polyethylene glycols (PEGs) as carriers of cytostatic compounds (Topchieva, I. N. (1990), Polym. Sci. USSR 32, 833-851; Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications (1992), Ed. J. M. Harris, Plenum Press, New York). PEGs are, due to their bio-compatibility, their good water-solubility and synthetic divergence, very suitable for the development of therapeutic polymer conjugates. In recent years, PEGs have been conjugated mainly with medically significant proteins and enzymes (Overview in Topchieva, I. N. (1990), Polym. Sci. USSR, 32, 833-851). The production of chemoimmuno-conjugates and conjugates of proteins or of polymers occurs generally either through direct coupling of carrier substance and active substance or with the help of spacer groups, so-called homo- or heterobifunctional reagents. Until now, mainly the method of direct coupling has been used which, however, often leads to polymeric products and not-unequivocally-defined conjugates. Recently, several chemoimmuno-conjugates (European Patent Application EP 91-117535 911615, European Patent Application EP 90-109268 900516, PCT International Patent Application WO 90-CA251 900809, British (UK) Patent Application GB 83-5104 830224 and European Patent Application EP 89-102370 890210), which were produced using specific bifunctional reagents, were suggested as cytostatically effective media. Furthermore, from DE 41 22 210 A1, conjugates of tumor-active compounds with transferrin or albumin are known, wherein the tumor-active compound is activated with N-hydroxy succinimide and carbodiimide and the thus-obtained mixture is directly coupled to the carrier protein.
It has now been found that conjugates of transferrin, albumin and polyethylene glycol, consisting of transferrin, albumin and polyethylene glycol, with a mass of between 5000 and 200000 Da and, at least, one cytostatic compound derivatized through compounds of maleinimide or N-hydroxysuccinimide, have a tumor-inhibiting effectiveness which is equal or higher than that of the cytostatic compound. Suitable for the production of these conjugates of protein or polymer are cytostatic compounds such as the anthracyclines, doxorubicin, daunorubicin, epirubicin, idarubicin and mitoxandrone, the alkylates, chloroambucil and melphalan, the antimetabolites, methotrexate, 5-fluorouracyl, 5xe2x80x2-desoxy-5-fluorouridine and thioguanine, the taxoides, paclitaxel and docetaxel, the camptothecins, topotecan and 9-aminocamptothecin, the podophyllotoxin derivatives, etoposide, teniposide and mitopodoside, the vinca alkaloids, vinblastine, vincristine, vindesine and vinorelbine and a compound of the general I, II, III or IV: 
n=0-6, X=xe2x80x94NH2, xe2x80x94OH, xe2x80x94COOH, xe2x80x94Oxe2x80x94COxe2x80x94Rxe2x80x94COR*, xe2x80x94NHxe2x80x94COxe2x80x94Rxe2x80x94COR*, wherein R is an aliphatic carbon chain with 1-6 carbon atoms or a substituted or unsubstituted phenylene group and R* H, phenyl, alkyl with 1-6 carbon, and the amine functions are provided with a protective group such as the tert.-butyloxycarbonyl protective group, which were derivatized with a compound of maleinimide or N-hydroxysuccinimide. In doing so, the cytostatic compounds are, as a rule, reacted with a maleinimide compound or N-hydroxysuccinimide compound which has at least one functional group which is suitable for binding to the cytostatic, such as an amino, hydroxy, carbonic acid, carbonic acid chloride, sulfonic acid, sulfonic acid chloride, acid hydracide, or hydrazino, oxycarbonyl chloride, aldehyde or keto group, so that maleinimide derivatives or N-hydroxysuccinimide ester derivatives of cytostatic compounds are prepared, wherein the chemical linkage between the maleinimide compound and cytostatic compound occurs through an amide, ester, imine, hydrazone, carboxyl hydrazone, oxycarbonyl, acetal or ketal bond. In the maleinimide or N-hydroxysuccinimide compounds which are obtained from the compounds of the formulas I-IV, the cytostatic cis-configured platinum unit is introduced subsequently, that is, the corresponding platinum(II)-complexes are obtained, after removal of the protective group, either through reaction with a tetrachloroplatinate salt or with cis-[PtA2B] (A=halogen, B=(NH3)2, ethylene diamine, propane diamine, 1,2-diaminocyclohexane).
Through reacting the derivatized cytostatic compounds with native or thiolated transferrin or albumin or with hetero- or homobifunctional PEGs with a mass of between 5000 and 200 000 Daxe2x80x94Overview 1:
conjugates of proteins or polymers are prepared which are produced simply and effectively, having a high purity, have an excellent water-solubility in comparison to several of the original cytostatic compounds, are stable formulations in a physiologic buffer and which have an in vitro anti-proliferation effectiveness against human tumor cells which is equal to or better than that of the unbound cytostatics. Furthermore, the conjugates exhibit a equal good or improved anti-tumor effectiveness in vivo and an improved tolerability. The conjugates of protein or polyethylene glycol realized through such couplings, which are very suitable for a selective treatment of cancer diseases, are object of the invention and are described in the following.
The method for the synthesis of conjugates of protein or polyethylene glycol occurs in the conjugates with maleinimide derivatives in four steps (Steps 1 to 4), in the conjugates with N-hydrosuccinimide ester derivatives in three steps (Steps 1, 2 and 4):
Step 1: Synthesis of maleinimide or N-hydroxysuccinimide compounds
Step 2: Synthesis of maleinimide derivatives or N-hydroxysuccinimide ester derivatives of cytostatic compounds
Step 3: Thiolation of the carrier protein
Step 4: Coupling of the cytostatic compound obtained in Step 2 to native or thiolated carrier protein or to a PEG shown in Overview 1.
Step 1: Synthesis of Maleinimide or N-hydroxysuccinimide Compounds
The maleinimide compounds are generally produced according to one of the following two methods:
In the first method, maleic acid anhydride is reacted with an aliphatic amino compound H2Nxe2x80x94Rxe2x80x94Y, wherein R is an aliphatic C-chain with 1-6 carbon atoms or a substituted or unsubstituted benzyl group and Y=xe2x80x94OH, xe2x80x94COOH, xe2x80x94SO3H, xe2x80x94CH(OC2H5)2, R* xe2x80x94Cxe2x95x90O, R*=phenyl or alkyl group with 1 to 6 carbon atoms, to yield the corresponding maleaminic acid and subsequently with triethylamine (Et5N) in an amount of up to two equivalents in non-aqueous toluol under azeotropic removal of the water obtained to yield the corresponding maleinimide compound. The further derivatization of the Y group occurs by reacting the xe2x80x94COOH or the xe2x80x94SO3H group with oxalyl chloride or thionyl chloride to yield the corresponding acid chlorides, by reacting the hydroxyl group with bis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonyl chlorides, by reacting the acetal group xe2x80x94CH(OC2H5,)2 to yield the corresponding aldehyde with the help of acid-catalytic cleavage such as, for example, through p-toluol sulfonic acid, sulfuric acid or acidic silica gel, and by reacting acid chloride with N-(tert.-butoxycarbonyl)-alcohol amine or N-(tert.-butoxycarbonyl)-alcohol hydrazine and subsequent cleavage with trifluoro acetic acid or hydrogen chloride (HCl) in ether, tetrahydrofuran (THF) or dioxan yielding the corresponding amino or hydrazino compounds, respectively.
In the second method, maleic acid anhydride is reacted with an aromatic amino compound H2Nxe2x80x94Rxe2x80x94Y, wherein R is a substituted or unsubstituted phenylene group and Y=xe2x80x94OH, xe2x80x94COOH, xe2x80x94SO3H, R* xe2x80x94Cxe2x95x90O, R*=phenyl or alkyl group with 1 to 6 carbon atoms, to yield the corresponding maleaminic acid and subsequently with acetic acid anhydride and anhydrous sodium acetate to yield the corresponding maleinimide compound. The further derivatization of the group Y occurs by reacting the xe2x80x94COOH or the xe2x80x94SO3H group with oxalyl chloride or thionyl chloride to yield the corresponding acid chlorides, by reacting the hydroxyl group with bis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonyl chlorides, by reacting the acid chloride to yield the corresponding aldehydes with the help of LiAl[OC(CH3)3]3H in THF, by reacting the acid chloride in THF or ethyl acetate with t-buytlcarbazate and subsequent cleavage with trifluoroacetic acid or HCl in ether, THF or dioxan to yield the corresponding acid hydrazides and by reacting the acid chlorides with N-(tert.-butoxycarbonyl)-alcohol amine or N-(tert.-butoxycarbonyl)-alcohol hydrazine and subsequent cleavage with trifluoroacetic acid or HCL in ether, THF or dioxan to yield the corresponding amino or hydrazino compounds.
The maleinimide compounds of the general formulas VI and VII are produced by reacting the maleinimide compounds obtained in the above-mentioned methods, in which Y is xe2x80x94COxe2x80x94NHNH2 or xe2x80x94COR* with n=1-6 and R*=H, phenyl, alkyl with 1-6 carbon atoms, with carbonyl compounds of the general formula Oxe2x95x90CR*xe2x80x94Rxe2x80x94Y or with acid hydrazide of the general formula Yxe2x80x94Rxe2x80x94R*COxe2x80x94NHxe2x80x94NH2 in anhydrous THF, methanol or ethanol with the optional addition of toluene-p-sulfonic acid or trifluoroacetic acid. A further derivatization of group Y occurs by reacting the COOHxe2x80x94 or the SO3H group with oxalylchloride or with thionyl chloride to yield the corresponding acid chlorides, by reacting the hydroxy group with bis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonyl chlorides.
The bismaleinimide compounds of the general formula XI, wherein two maleinimide compounds are connected by a group Z, which represents a diaminoalkane, dihydroxyalkane, dihydrazinoalkane or carboxylic acid dihydrazide compound, so that two maleinimide compounds are connected with one another via two amide, ester, imine, hydrazone or carboxylhydrazone bonds, are produced from the above-mentioned maleinimide compounds, wherein the synthesis of the compounds connected by the amine bonds occurs by reacting the acid chloride of the maleinimide compounds with diaminoalkane compounds NH2xe2x80x94(CH2)nxe2x80x94NH2, n=2-12, in THF or ethyl acetate with the optional addition of Et3N, the synthesis of the compounds connected by an ester bond by reacting the acid chloride of the maleinimide compounds with dihydroxy compounds HOxe2x80x94(CH2)nxe2x80x94OHxe2x80x94, n=2-12, in THF or or ethyl acetate with the optional addition of Et3N, the synthesis of the compounds connected by an imine bond by reacting the aldehydes or ketones of the maleinimide compounds with diaminoalkane compounds NH2xe2x80x94(CH2)nxe2x80x94NH2, n=2-12, in anhydrous THF, methanol or ethanol with the addition of toluene-p-sulfonic acid or trifluoroacetic acid and the synthesis of the compounds connected by a hydrazone or carboxyl hydrazone bond occurs by reacting the aldehydes or ketones of the maleinimide compounds with dihydrazinoalkane compounds NH2xe2x80x94NHxe2x80x94(CH2)nxe2x80x94NHxe2x80x94NH2 or carboxylic acid dihydrazines H2 Nxe2x80x94NHxe2x80x94COxe2x80x94(CH2) nxe2x80x94COxe2x80x94NHxe2x80x94NH2, n=2-12, in anhydrous THF, methanol or ethanol with the addition of toluene-p-sulfonic acid or trifluoroacetic acid.
The N-hydroxysuccinimide compounds are produced in general by reacting N-hydroxysuccinimide with Yxe2x80x94Rxe2x80x94COOH or with Yxe2x80x94Rxe2x80x94COCl, wherein R is a substituted or unsubstituted phenylene group, Y=xe2x80x94OH, xe2x80x94NH2, xe2x80x94COOxe2x80x94(CH2)nxe2x80x94OH, xe2x80x94CONHxe2x80x94(CH2)nNHBOC, xe2x80x94NHNHBOC, xe2x80x94COOxe2x80x94(CH2)nxe2x80x94NHNHBOC, xe2x80x94SO3H, xe2x80x94SO2xe2x80x94NHNHBOC, xe2x80x94CHO, xe2x80x94COR*, xe2x80x94COxe2x80x94NHNHBOC with n=1-6 and R*=H, phenyl, alkyl with 1-6 carbon atoms and BOC is the tert.-butyloxycarbonyl protective group, to yield the corresponding N-hydroxysuccinimide ester compound. In so doing, the reaction starting with Yxe2x80x94Rxe2x80x94COOH is performed in an anhydrous solvent, preferably dichloromethane, acetonitril or THF with the addition of dimethylaminopyridine (DMAP) and a condensation agent, as a rule, N,Nxe2x80x2-dicyclohexylcarbodiimide (DCC) or N-cyclohexyl-Nxe2x80x2-(2-morpholinoethyl)-carbodiimide metho-p-toluol sulfonate (CMC), to yield the corresponding succinimide ester derivatives. If the acid chloride Yxe2x80x94Rxe2x80x94COCl, which is obtained through chlorination with acid halogenation reagents such as, for example, with oxalylchloride or thionyl chloride, is employed, then the reaction with N-hydroxysuccinimide occurs preferably in anhydrous THF, acetonitril or ethyl acetate.
The BOC protective group can subsequently be removed with trifluoroacetic acid or HCl in ether or dioxan, so that the corresponding amino or hydrazino compounds as well as the acid hydrazides are obtained as trifluoroacetate or hydrochlorides. A further derivatization of group Y occurs by reacting the hydroxy group with bis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonyl chlorides. N-Hydroxysuccinimide compounds of the general formulas IX and X are produced by reacting the hydroxysuccinimide compounds obtained in the above-mentioned methods, in which Y is xe2x80x94COxe2x80x94NHNH2 or xe2x80x94COR* with n=1-6 and R*=H, phenyl, alkyl with 1-6 carbon atoms, with carbonyl compounds of the general formula Oxe2x95x90CR*xe2x80x94Rxe2x80x94Y or with acid hydrazides of the general formula Yxe2x80x94Rxe2x80x94R*COxe2x80x94NHxe2x80x94NH2 in anhydrous THF, methanol or ethanol with the optional addition of toluene-p-sulfonic acid or trifluoroacetic acid. The further derivatization of group Y occurs by reacting the COOH or the SO3H group with oxalylchloride or with thionyl chloride to yield the corresponding acid chlorides, by reacting the hydroxy group with bis-(trichloromethyl)-carbonate to yield the corresponding oxycarbonyl chlorides.
The isolation of the above-mentioned maleinimide and N-hydroxysuccinimide ester compounds occurs either through crystallization, through silica gel column chromotography or through preparative HPLC or LPLC on a diol column, as is described in the examples below.
Step 2: Maleinimide Derivatives or N-hydroxysuccinimide Derivatives of Cytostatic Compounds
Suitable for the reaction with the maleinimide and N-hydroxysuccinimide compounds obtained in Step 1 are the cytostatic compounds mentioned in claims 1 to 3. These cytostatic compounds are reacted with the maleinimide and N-hydroxysuccinimide ester compounds described in Step 1, so that the maleinimide derivatives and N-hydroxysuccinimide ester derivatives of cytostatic compounds are provided, wherein the chemical linkage between maleinimide compound or N-hydroxysuccinimide ester compound and cytostatic compound occurs through an amide, ester, imine, hydrazone, carboxyl hydrazone, acetal or ketal bond.
In the case of the anthracyclines, doxorubicin, daunorubicin, epirubicin or idarubicin, the synthesis in detail through the reaction with acids or acid chlorides, listed in Step 1, of maleinimide or N-hydroxysuccinimide of formulas V to X to yield the corresponding anthracycline-maleinimide derivatives or corresponding anthracycline-hydroxysuccinimide derivatives in a solvent, preferably dimethylformamide (DMF) or THF with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via the 3xe2x80x2xe2x80x94NH2 group of the amino sugar of anthracycline as amide an bond,
or through reaction with the aldehydes or ketones of maleinimide or N-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferably DMF, methanol or ethanol, with the optional addition of an acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein the coupling occurs via the 3xe2x80x2xe2x80x94NH2 group of the amino sugar of anthracycline as an imine bond,
or through the reaction with the amines of maleinimide or N-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferably DMF, methanol or ethanol, with the addition of an acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein the coupling occurs via the C13-keto position of the anthracycline as an imine bond,
or through the reaction with the acid hydrazides of maleinimide or N-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferably DMF, methanol or ethanol, with the addition of an acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein the coupling occurs via the C13-keto position of the anthracycline as a carboxyl or sulfonyl hydrazone bond.
In the case of mitoxandron, the synthesis is performed in detail through reaction with the acids or acid chlorides of maleinimide or N-hydroxysuccinimide compounds, listed in Step 1, of formulas V to X to yield the corresponding mitoxandron-maleinimide derivatives or mitoxandron-hydroxysuccinimide derivatives in a solvent, preferably DMF or THF with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via, at least, one of the aliphatic HO-groups of the mitoxandron as an ester bond,
or through reaction with the aldehydes or ketones of maleinimide or N-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferably THF, methanol or ethanol with the optional addition of an acid, as a rule, trifluoroacetic acid or toluene-p-sulfonic acid, wherein the coupling occurs via, at least, one of the aliphatic HO-groups of the mitoxandron as an acetal or ketal bond.
In the case of the alkylating agents, chloroambucil and melphalan, the synthesis is performed in detail through reaction of chloroambucil or melphalan with the hydroxy compounds of maleinimide or N-hydroxysuccinimide compounds listed in Step 1 in a solvent, preferably DMF, dichloromethane or THF with the addition of DMAP and a condensation agent, as a rule, DCC or CMC, to yield the corresponding chloroambucil or melphalan-maleinimide derivatives or chloroambucil or melphalan-hydroxysuccinimide derivatives, respectively, wherein the coupling occurs via the COOH group of chloroambucil or melphalan as an ester bond,
or through reaction of chloroambucil or melphalan, respectively, with acid halogenation reagents such as oxalylchloride or thionyl chloride, to yield the corresponding acid chlorides and subsequent reaction of acid chlorides in THF or ethyl acetate with t-alkylcarbazates, as a rule, tert.-butylcarbazates, or with optional addition of a tertiary base, as a rule, Et3N, or through reaction of chloroambucil or melphalan in DMF, THF or ethyl acetate with t-alkylcarbazates, as a rule, DCC or CMC, and subsequent cleavage of the thus-obtained products with acids, as a rule, trifluoroacetic acid or HCl in ether, THF or dioxan, to yield the corresponding acid hydrazides of chloroambucil or melphalan, respectively, which, in turn, are reacted with one of the aldehydes or ketones, listed in Step 1, of maleinimide or N-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol or ethanol with the addition of an acid, as a rule, trifluoroacetic acid or toluene-p-sulfonic acid, to yield the corresponding maleinimide or N-hydroxysuccinimide carboxyl hydrazone derivatives of chloroambucil or melphalan, respectively.
In the case of 5-fluorouracil, the synthesis occurs in detail through reaction with the acid chlorides, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds to yield the corresponding maleinimide or N-hydroxysuccinimide derivatives of 5-fluorouracil in a solvent, preferably THF, with the optional addition of a tertiary base, as a rule, Et3N, wherein the coupling occurs via the 1N- or 3N-position of 5-fluorouracil as an acid amide bond,
or through the reaction with the oxycarbonyl chlorides, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds to yield the corresponding maleinimide or N-hydroxysuccinimide derivatives of 5-fluorouracil in a solvent, preferably THF, with the optional addition of a tertiary base, as a rule, Et3N, wherein the coupling occurs via the 1N- or 3N-position of 5-fluorouracil as an oxycarbonyl bond,
or through the reaction of 5-fluorouracil with formaldehyde and the carboxylic acids and sulfonic acids, listed in Step 1, to yield the corresponding maleinimide or N-hydroxysuccinimide derivatives of 5-fluorouracil in a solvent, preferably dichloromethane or THF, with the addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via the 1N- or 3N-position of 5-fluorouracil as a carbamoyloxymethyl bond.
In the case of 5xe2x80x2-desoxy-5-fluorouridine, the synthesis is performed in detail through reaction with the acid chlorides, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds of formulas V to X to yield the corresponding maleinimide or N-hydroxysuccinimide derivatives of 5xe2x80x2-desoxy-5-fluorouridine in a solvent, preferably THF, with the optional addition of a tertiary base, as a rule, Et3N, wherein the coupling occurs via the 2xe2x80x2xe2x80x94HO or 3xe2x80x2xe2x80x94HO group of 5xe2x80x2-desoxy-5-fluorouridine as an ester bond,
or through the reaction with the aldehydes or ketones, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds in a solvent, preferably THF, methanol or ethanol, with the addition of an acid, as a rule, trifluoroacetic acid or toluene-p-sulfonic acid, wherein the coupling occurs via the 2xe2x80x2xe2x80x94HO and/or 3xe2x80x2xe2x80x94HO group of 5xe2x80x2-desoxy-5-fluorouridine as an acetal or ketal bond.
In the case of thioguanine, the synthesis is performed in detail through the reaction with the acid chlorides, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds of formulas V to X to yield the corresponding maleinimide or N-hydroxysuccinimide derivatives of thioguanine in a solvent, preferably DMF, with the optional addition of a tertiary base, as a rule, Et3N, wherein the coupling occurs via the H2N group of thioguanine as an amide bond,
or through the reaction with the aldehydes or ketones, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol or ethanol, with the addition of an acid, as a rule, trifluoroacetic acid or toluene-p-sulfonic acid, wherein the coupling occurs via the H2N group of thioguanine as an imine bond.
In the case of methotrexate, the synthesis is performed in detail through the reaction of methotrexate with the hydroxy or amino compounds of maleinimide or N-hydroxysuccinimide compounds, listed in Step 1, in a solvent, preferably DMF or dimethylsulfoxide with the addition of DMAP and a condensation agent, as a rule, DCC or CMC, to yield the corresponding methotrexate-maleinimide derivatives or methotrexate-hydroxysuccinimide derivatives, respectively, wherein the coupling occurs either via the xcex1-COOH group or xcex3-COOH group or via both COOH groups of methotrexate as an ester or amide bond,
or through the reaction of methotrexate with alkylcarbazates, as a rule, t-butylcarbazate, in a solvent, preferably DMF or dimethyl sulfoxide with the addition of DMAP and a condensation agent, as a rule, DCC or CMC, and subsequent cleavage with acids, as a rule, trifluoroacetic acid or HCl in ether, THF or dioxan, to yield the corresponding acid hydrazides of methotrexate, wherein the acid hydrazide group was introduced at either the xcex1-COOH group or xcex3-COOH group or at both COOH groups of methotrexate, and the thus-obtained acid hydrazide derivatives of methotrexate are reacted now with one of the aldehydes or ketones, listed is Step 1, of the N-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol, THF or ethanol, with the addition of an acid, as a rule, trifluoroacetic acid or toluene-p-sulfonic acid, to yield the corresponding N-hydroxysuccinimide carboxylhydrazone derivatives of methotrexate.
In the case of the taxoides, paclitaxel and docetaxel, the synthesis is performed in detail through the reaction with the acids or acid chlorides, listed in Step 1, of maleinimide or N-hydroxysuccinimide compounds of formulas V to X to yield the corresponding taxoid-maleinimide derivatives or taxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF or THF with the optional addition of a tertiary base, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via the C7xe2x80x94 or C10xe2x80x94OH group of the taxoid as an ester bond,
or through the reaction with the amines or hydrazines, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol or ethanol, with the addition of an acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein the coupling occurs via the C9-keto position of the taxoid as an imine or hydrazone bond,
or through the reaction with the acid hydrazides, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds in a solution, preferably DMF, methanol or ethanol, with the addition of an acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein the coupling occurs via the C9-keto position of the taxoid as a carboxyl or sulfonyl hydrazone bond.
In the case of the camptothecines, topotecan or 9-aminocamptothecine, the synthesis is performed in detail through the reaction with the acids or acid chlorides, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds of formulas V to X to yield the corresponding taxoid maleinimide derivatives or taxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF or THF with the optional addition of a tertiary base, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via the C10xe2x80x94OH group of the topotecan as an ester bond or via the C9xe2x80x94NH2 group of the 9-aminocamptothecin as an amide bond,
or through the reaction of 9-aminocamptothecin with the aldehydes or ketones, listed in Step 1, of of the maleinimide or N-hydroxysuccinimide compounds in a solution, preferably DMF, methanol or ethanol, with the optional addition of an acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, wherein the coupling occurs via the C9xe2x80x94NH2 group as an imine bond.
In the case of the podophyllotoxin derivatives, etoposide, teniposide and mitopodozide, the synthesis is performed in detail through reaction with the acids or acid chlorides, listed in Step 1, of maleinimide or N-hydroxysuccinimide compounds of formulas V to X to yield the corresponding taxoid-maleinimide derivatives or taxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF, dichloromethane or THF with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via one of the aliphatic HO-groups of the podophyllotoxin derivative as an ester bond.
In the case of the vinca alkaloids, vinblastine, vincristine, vindesine and vinorelbine, the synthesis occurs in detail through the reaction with the acids or acid chlorides, listed in Step 1, of maleinimide or N-hydroxysuccinimide compounds of formulas V to X to yield the corresponding taxoid-maleinimide derivatives or taxoid-hydroxysuccinimide derivatives in a solvent, preferably DMF, dichloromethane or THF with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, wherein the coupling occurs via one of the aliphatic HO-groups of the vinca alkaloid as an ester bond.
In the case of maleinimide or N-hydroxysuccinimide derivatives of the cis-configured platinum(II)-complexes, the synthesis occurs in detail through the reaction of the corresponding amino compounds H2Nxe2x80x94CH2CH2xe2x80x94NHxe2x80x94(CH2)nxe2x80x94X, (H2Nxe2x80x94CH2)2CHxe2x80x94(CH2)nxe2x80x94X or H2Nxe2x80x94CH2CH(NH2)xe2x80x94(CH2)nxe2x80x94X (general formulas I, II and III), wherein one or two of the primary or secondary amino groups has been protected with a BOC group (reaction with bis-tert.-butyloxy carbonyl anhydride) and X is xe2x80x94NH2 or xe2x80x94OH, with the acids or acid chlorides, listed in Step 1, of maleinimide or N-hydroxysuccinimide compounds of the general formulas V-X in a solvent, preferably THF or ethyl acetate, with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, to yield the corresponding BOC-protected maleinimide or hydroxysuccinimide derivatives which then are converted by means of trifluoroacetic acid or HCl in ether, THF or dioxan through cleavage-off of the BOC group into the corresponding trifluoroacetate or hydrochloride and finally through reaction with a tetrachloro-platinate(II) salt, preferably potassium tetrachloro-platinate(II), in water, salt buffers, DMF, DMF/water mixtures, THF/water mixtures or DMF/methanol mixtures, into the corresponding platinum(II)-complexes, wherein the coupling occurs via the terminal HO group as an ester bond or via the terminal H2N group as an acid amide bond, or through the reaction of the corresponding amino compounds H2Nxe2x80x94CH2CH2xe2x80x94NHxe2x80x94(CH2)nxe2x80x94X, (H2Nxe2x80x94CH2)2CHxe2x80x94(CH2)nxe2x80x94X or H2Nxe2x80x94CH2CH(NH2)xe2x80x94(CH2)nxe2x80x94X (general formulas I, II and III), wherein one or two of the primary or secondary amino groups has been protected with a BOC group (reaction with bis-tert.-butyloxy carbonyl anhydride) and X is xe2x80x94NH2 or xe2x80x94OH, with compounds of the type HOOCxe2x80x94Rxe2x80x94COCR* or ClOCxe2x80x94Rxe2x80x94COCR* (R is an aliphatic carbon chain with 1-6 carbon atoms or a substituted or unsubstituted phenylene group, and R* is H, phenyl, alkyl with 1-6 carbon atoms) in a solvent, preferably THF or ethyl acetate, with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, to yield the corresponding BOC-protected maleinimide or hydroxysuccinimide derivatives which now have a further carbonyl function which, in the following, are reacted with the amines, acid hydrazides or hydrazines, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol or ethanol, with the addition of acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, to yield the corresponding imine, carboxylhydrazone or hydrazone derivatives, which then again are converted by means of trifluoroacetic acid or HCl in ether, THF or dioxan through cleavage-off of the BOC group into the corresponding trifluoroacetate or hydrochloride and finally through reaction with a tetrachloro-platinate(II) salt, preferably potassium tetrachloro-platinate(II), in water, salt buffers, DMF, DMF/water mixtures, THF/water mixtures or DMF/methanol mixtures, into the corresponding platinum(II)-complexes, or through the reaction of the corresponding amino compounds H2Nxe2x80x94CH2CH2xe2x80x94NHxe2x80x94(CH2)nxe2x80x94X, (H2Nxe2x80x94CH2)2CHxe2x80x94(CH2)nxe2x80x94X or H2Nxe2x80x94CH2CH(NH2)xe2x80x94(CH2)nxe2x80x94X (general formulas I, II and III), wherein one or two of the primary or secondary amino groups has been protected with a BOC group (reaction with bis-tert.-butyloxy carbonyl anhydride) and X is COOH or this carbonyl group was converted using acid halogenation reagents such as thionyl chloride or oxalyl chloride into the acid chloride, with compounds of the type HORxe2x80x94COCR* or H2Nxe2x80x94Rxe2x80x94COCR* (R is an aliphatic carbon chain with 1-6 carbon atoms or a substituted or unsubstituted phenylene group, and R* is H, phenyl, alkyl with 1-6 carbon atoms) in a solvent, preferably THF or ethyl acetate, with the optional addition of a tertiary base, as a rule, Et3N, or with the optional addition of DMAP and a condensation agent, as a rule, DCC or CMC, to yield the corresponding BOC-protected maleinimide or hydroxysuccinimide derivatives which now have a further carbonyl function which, in the following, are reacted with the amines, acid hydrazides or hydrazines, listed in Step 1, of the maleinimide or N-hydroxysuccinimide compounds in a solvent, preferably DMF, methanol or ethanol, with the addition of acid, as a rule, toluene-p-sulfonic acid or trifluoroacetic acid, to yield the corresponding imine, carboxylhydrazone or hydrazone derivatives, which then again are converted by means of trifluoroacetic acid or HCl in ether, THF or dioxan through cleavage-off of the BOC group into the corresponding trifluoroacetate or hydrochloride and finally through reaction with a tetrachloro-platinate(II) salt, preferably potassium tetrachloro-platinate(II), in water, salt buffers, DMF, DMF/water mixtures, THF/water mixtures or DMF/methanol mixtures, into the corresponding platinum(II)-complexes.
In the case of maleinimide or N-hydroxysuccinimide derivatives with malonic acid derivatives of the general formula IV (HOOC)2xe2x80x94CHxe2x80x94(CH2)nxe2x80x94X to the cis-configured platinum(II)-complexes, the synthesis occurs analogous to the above-described complexes, wherein the platinum(II)-complexes are obtained by reacting the maleinimide or N-hydroxysuccinimide derivatives with malonic acid derivatives with cis-[PtA2B] (A=halogen, preferably Cl or J, B=(NH3)2, ethylene diamine, propane diamine, 1,2-diaminocyclohexane) to yield the corresponding platinum(II)-complexes in a solvent, such as water salt buffers, DMF, DMF/water mixtures, THF/water mixtures or DMF/methanol mixtures with the addition of a hydroxide solution, preferably aqueous KOH. The reaction can optionally be carried out in the presence of silver nitrate (AgNO3) or silver sulfate (Ag2SO4). The platinum complex is obtained through crystallization or through addition of a solvent, preferably diethylether or THF.
The isolation of the above-mentioned maleinimide or N-hydroxysuccinimide cytostatic compounds, respectively, occurs either through crystallization, through silica gel column chromatography or through preparative HPLC or LPLC on a reverse-phase (C8 or C18) or diol column, as is described in the examples below.
Step 3: Thiolation of the Carrier Protein
Sulfohydryl groups (HS groups) are introduced through reaction of the carrier protein with a thiolation reagent, preferably iminothiolan, into human serum transferrin and serum albumin. The thiolation occurs in a salt buffer, as a rule, in 0.1 M sodium borate, 0.15 M NaCl, 0.001 M EDTAxe2x80x94pH=8.0, with an excess of thiolation reagent (2- to 100-fold excess) and subsequent gel filtration (for example, Sephadex(copyright) G10 of G25) with a salt buffer such as 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0-7.5 or 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0-7.5. The concentration of protein after completed gel filtration is determined through the extinction coefficient at 280 nm and is, as a rule, in the range of between 1.0xc3x9710xe2x88x924 and 5.0xc3x9710xe2x88x923 M. The number of the introduced HS groups is determined with Ellmann""s reagent at 412 nm. Through variation of the reaction conditions, 1 to 30 HS groups can be introduced on the average. The thiolated transferrin or albumin solution is employed directly for the synthesis of the conjugates.
Step 4: Coupling of the Cytostatic Maleinimide or N-hydroxysuccinimide Compounds to the Native or Thiolated Carrier Protein or to a Polyethylene Glycol Shown in Overview 1
For the coupling of the cytostatic maleinimide or N-hydroxysuccinimide compounds to PEGs, PEGs are employed which have one or two HOxe2x80x94, HSxe2x80x94 or H2N groups and a mass of between 5,000 and 200,000 Da, preferably between 20,000 and 70,000 Da. Corresponding compounds are not commercially available. In the following, polyethylene glycols having one or two HS groups are shortened with HS-PEG or HS-PEG-SH, and the PEGs having one or two H2N groups are shortened with H2N-PEG or H2N-PEG-NH2.
Coupling of the cytostatic maleinimide derivatives to the thiolated carrier protein or to HS-PEG, HS-PEG-SH, H2N-PEG or H2N-PEG-NH2: The cytostatic maleinimide derivatives (see Step 2) are reacted with thiolated transferrin, albumin (see Step 3), HS-PEG, HS-PEG-SH, H2N-PEG or H2N-PEG-NH2 at room temperature. In so doing, to the thiolated protein, HS-PEG, HS-PEG-SH, H2N-PEG or H2N-PEG-NH2, which is present in a degassed salt buffer such as 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0 to 7.5 or 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0 to 7.5, an approximately 1.1- to 10-fold excess of the cytostatic maleinimide derivative is added (in terms of the number of available HS groups in the protein or PEG), dissolved in a minimal amount of solvent, as a rule, DMF, dimethylsulfoxide, water, ethanol, methanol, acetonitril or THF (approximately 1 to 10% of the volume of the thiolated sample). After approximately 5 to 120 minutes, the solution is centrifuged, and the formed protein conjugate or PEG conjugate is separated off through subsequent gel filtration (for example, Sephadex(copyright) G10, G25 or LH20) in a degassed salt buffer such as 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0-7.5, 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0-7.5 or 0.1-0.2 M NaHCO3, or in methanol or THF, from the excess cytostatic maleinimide derivative. It can be advantageous to dilute the thiolated protein solution prior to the addition of the maleinimide derivative with a salt buffer and to add the maleinimide derivative, which is dissolved in a minimal amount of solvent, and subsequently to concentrate the solution after 5-20 minutes with a customary commercial concentrator and to isolate the protein conjugate, as described above. Further, the solution of the thus-obtained protein conjugate or of the PEG conjugate can be concentrated with a customary commercial concentrator or the solvent removed under a high vacuum.
Coupling of the cytostatic N-hydroxysuccinimide derivatives to the native carrier protein or to HO-PEG, HO-PEG-OH, H2N-PEG or H2N-PEG-NH2: The cytostatic N-hydroxysuccinimide derivatives (see Step 2) are reacted with transferrin, albumin, HO-PEG, HO-PEG-OH, H2N-PEG or H2N-PEG-NH2 at room temperature. In so doing, to the protein, HO-PEG, HO-PEG-OH, H2N-PEG or H2N-PEG-NH2, which is located in a degassed salt buffer such as 0.025 M sodium borate, 0.15 M NAClxe2x80x94pH 6.0 to 8.0 or 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0 to 8.0, an approximately 1.1- to 50-fold excess of the cytostatic N-hydroxysuccinimide derivative is added, dissolved in a minimal amount of solvent, as a rule, DMF, dimethylsulfoxide, water, ethanol, methanol, acetonitril or THF (approximately 1 to 10% of the volume of the thiolated sample). After approximately 5 minutes to 48 hours, the solution is centrifuged, and the formed protein conjugate or PEG conjugate is separated off through subsequent gel filtration (for example, Sephadex(copyright) G10, G25 or LH20) in a degassed salt buffer such as 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0-7.5, 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0-7.5 or 0.1-0.2 M NaHCO3, or in methanol or THF, from the excess cytostatic N-hydroxysuccinimide derivative. The solution of the thus-obtained protein conjugate or of the PEG conjugate can be concentrated with a customary commercial concentrator or the solvent removed under a high vacuum.
The number of the cytostatic maleinimide derivatives or N-hydroxysuccinimide derivatives bound to the carrier protein or to the polyethylene glycol is specified either through a photometric concentration determination at the absorbed wavelength of the cytostatic maleinimide or N-hydroxysuccinimide derivative (typically between 220 and 600 nm) and/or through colorimetric determination which, in the case of the conjugates of chloroambucil and melphalan, is performed with the aid of the NBP test (Epstein, J., Rosenthal, R. W., Ess, R. J. Anal. Chem. (1955), 27, 1435-1439), in the case of the conjugates of 5-fluorouracil and 5xe2x80x2-desoxy-5-fluorouridine with the aid of an assay according to Habib (Habib, S. T., Talanta (1981), 28, 685-87) and in the case of the conjugates of the cis-configured platinum(II)-analogues with the aid of a determination according to Gonias and Pizzo (Gonias, S. L., Pizzo, S. V. J.Biol.Chem. (1982), 258, 5764-5769) or according to atomic absorption spectroscopy (AAS).
On the average, through the above-described way, approximately 1-30 molecules of the cytostatic compound is bound to one molecule of protein or 1-2 molecules of the cytostatic compound to one molecule of the PEGs. The purity of the protein conjugate or the PEG conjugate is checked through HPLC with the aid of an analytical column (Bio-Sil SEC 250, (300 mmxc3x977.8 mm) from Bio-RAD, mobile phase: as a rule, 0.15 M NaCl, 0.01 M NaH2PO4, 5% of CH3CNxe2x80x94pH 7.0 or Nucleogel(copyright) aqua-OH 40 or 60, from Macherey and Nagel, mobile phase: as a rule, 0.1 M NaCl, 0.004 M NaH2PO4, 30% methanolxe2x80x94pH 7.0). In so doing, the transferrin, albumin and polyethylene glycol conjugates exhibit a purity of  greater than 90%.
The protein conjugates or PEG conjugates can be stored in a dissolved form at 0-5xc2x0 C., in frozen form at T=xe2x88x9220xc2x0 C. or xe2x88x9278xc2x0 C. Furthermore, it is possible to lyophilize the solution of the conjugate and store the lyophilisate at +5 to xe2x88x9278xc2x0 C.
Object of the invention are also such chemoimmuno-conjugates consisting of albumin which, according to one of the claims 1 to 3, is loaded with approximately two to thirty equivalents of a cytostatic compound and is conjugated, with a protein, for example, with transferrin or with a monoclonal antibody which is directed against a tumor-associated antigen, preferably, however, with transferrin, via one of the bismaleinimide compounds mentioned in claim 3 or via an aliphatic or aromatic bismaleinimide compound. In so doing, approximately 80-90% of the thiol groups introduced into albumin with the cytostatic maleinimide derivative, dissolved in a minimal amount of solvent, as a rule, DMF, dimethylsulfoxide, ethanol, methanol, acetonitril or THF (approximately 1-10% of the volume of the thiolated sample) are reacted and, after approximately 5 to 60 minutes, a 1.5- to 20-fold excess of the bismaleinimide compound, dissolved in a minimal amount of solvent, as a rule, DMF, dimethylsulfoxide, ethanol, methanol, acetonitril or THF (approximately 1-10% of the volume of the thiolated sample) is added. After approximately 5 to 20 minutes, the solution is centrifuged, and the formed protein conjugate separated off through subsequent gel filtration (for example, Sephadex(copyright) G10 or G25) in a salt buffer, as a rule, 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0-7.5 or 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0-7.5, from the cytostatic maleinimide derivative and the bismaleinimide compound. Then the thus-modified albumin conjugate is reacted with one of the proteins mentioned above which contains approximately 1.5 thiol groups on the average, and the resulting chemoimmuno-conjugate which now consists of an albumin molecule loaded with a cytostatic compound and of the above-mentioned protein, is isolated with the aid of a Superdex-200 column (company: Pharmacia) or a hydroxyl apetite column (company: Pharmacia or Bio-Rad) in a salt buffer, as a rule, 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.5-8.0, 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.5-8.0.
It is also possible to, first, react one of the above-mentioned proteins, which contains 1.5 thiol groups on the average, with a 1.5- to 10-fold excess of the bismaleinimide compound and then to separate the formed protein conjugate through subsequent gel filtration (for example, Sephadex(copyright) G10 or G25) in a degassed salt buffer such as 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0-7.5, 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0-7.5 or 0.1-0.2 M NaHCO3, from the bismaleinimide compound, and then to react the thus-modified protein conjugate, which now contains 1.5 equivalents of the bismaleinimide compound on the average, with the modified albumin conjugate, wherein approximately 80-90% of the thiol groups introduced into the albumin have already been reacted with a cytostatic maleinimide derivative. The resulting chemoimmuno-conjugate which now consists of an albumin molecule loaded with a cytostatic compound and of the above-mentioned protein, is isolated with the aid of a Superdex-200 column (company: Pharmacia) or a hydroxyl apatite column (company: Pharmacia or Bio-Rad) in a degassed salt buffer, such as, 0.025 M sodium borate, 0.15 M NaClxe2x80x94pH 6.0-8.0, 0.004 M phosphate, 0.15 M NaClxe2x80x94pH 6.0-8.0 or 0.1-0.2 M NaHCO3.
The following examples describe the invention in more detail without limiting it.